Cloning And Analysis Of The Senescence-associated Genes From Plants

Plant senescence is a highly regulated developmental process that ends with the programmed death of cells. Although a wide range of the senescence-associated genes (SAGs) have been identified and analyzed in a number of species, more novel SAGs need to be identified and characterized before the mechanism of senescence is fully understood. In this dissertation, the SAGs, eukaryotic translation initiation factor 5A (eIF5A) and yellow-leaf-specific gene 7 (YLS7),GcB5 were cloned and analyzed from wheat and Gymanadenia Conopsea R. Br, respectively. The results provided important information for study SAGs, and basic data of bacterial proteome for the regulation control and reconstruction of E. coli BL21, a gene engineering bacterium. The main results showed as follows:1. A full-length cDNA clone (TaeIF5A-l) was isolated by 5', 3'- RACE and two genomic sequences (TaeIF5A-2 and TaeIF5A-3) encoding eIF5A in wheat (Triticum aestivum) were also cloned. In addition, 9 partial DNA sequences of eIF5A gene were also isolation from another nine species of triticeae tribe.2. Phylogenetic analysis of eIF5A coding genes in general revealed that wheat eIF5A represents ancestral types of the gene family in plant. Phylogenetic analysis of ten species of triticeae tribe according to the second intron of eIF5A indicate that eIF5A evaluated in step with species3. Physical locating analysis show that TaeIF5A-2 was located on the chromosome 2BL using Langdon D-genomic disomic substitution lines and the Chinese Spring ditelosomic lines. According to the BLAST result of TaeIF5A-l on the wheat EST website (http://wheat.pw.usda.gov/wEST/blast), it is inferred that TaeIF5A-2 have three copies which lie on the 2AL, 2DL and 7BL.4. The expressive pattern of eIF5A at the mRNA level was researched by semi-quantitative RT-PCR, the results of down-regulated expression pattern in flag leaf and the lowest expression level in root indicated that TaeIF5A-1 has spatial and temporal difference and it was mainly involve in leaf development.5. Structure and organization of eEF5A analysis show that the wheat eIF5A genes were comprised of 6 extrons and 5 introns. The ATG start codon and the TAA stop codon locate on the extron 2 and extron 6, respectively. The identity between genomic sequences TaeIF5A-2 and TaeIF5A-3 was 82.3%. They share identical transcriptions of 636bp, in which have only 6 nucleotides difference. The intron 2 of the two sequences was larger and more variation than other introns. The introns 2 of TaeIF5A-2 and TaeIF5A-3 have 896bp and 672bp, respectively, and they share only 59% identity.6. According to the sequence of TaeIF5A-l, the expression vector pET28a:eIF5A was constructed. Prokaryotic expression indicated that the eIF5A of wheat expressed one approximate 18kD proteins the in E. coli and Western blot analysis confirmed that the expression proteins were translation initiation factor 5A.7. A modifying Oligo-capping method has been developed in this dissertation. Using this method, we successfully constructed a full-length cDNA library of Gymanadenia Conopsea R. Br. which contained 8×10⁵ independent clones with more than 96% were recombinant. Sequence analysis results indicated that more than 80% of the clones were full-length. Utilizing this cDNA library, we can isolate the senescence-associated genes (SAGs).8. A single cDNA clone, GcB5 (DQ407755) was obtained from the full-length enriched cDNA library by PCR with special primers of leaf senescence gene. Nucleotide sequences of GcB5 was determined to contain 1810 bp, and contain a 169-bp 5'-nocoding sequence and a 216-bp 3'-nocoding sequence, and it encoded putatively a 474 amino acid polypepitide with a calculated molecular mass of 53 kDa. GcB5 showed identities of 51.9% and 56.55% to the SAG of Arabidopsis thaliana YLS7 (AB047810) at nucleotide and amino acid level, respectively, suggest that GcB5 was a senescence-associated gene.9. The prokaryotic expression vector pET28a:GcB5 has also been constructed. Prokaryotic expression shows no distinct recombinant protein band can be observed in SDS-PAGE; the condition of E. coli cell growth affected by GcB5 was also examined on LB plates and monitored by measuring OD₆₀₀ of liquid cultures indicate that the GcB5 induced to inhibit growth and proliferation of E. coli, either directly or indirectly.10.In order to understand the mechanisms of GcB5-induced growth inhibition, proteomic strategy of 2-DE coupled with MALDI-TOF MS/MS were applied to investigate the proteins of E. coli related to growth inhibition. From the 2-DE separations, 99 spots representing 91 proteins associated with cell growth inhibition were identified. Among them, 55 proteins were involved in metabolism and biosynthesis; and three proteins were reported here to be antibiotic-resistance-related proteins; and moreover, twelve interesting and important proteins were resulting for cell division. These results suggested that expression of recombinant protein GcB5 resulted in rapid stress response and global effect on E. coli.11. Semi-quantitative RT-PCR was applied to the determination of changes in 16 genes (grpE, ompA, oxyR, mreB, pspA, cysK, ompF, clpX, hslU, pnp, ftsZ, minD, ahpC, yeaD, tolC and dacA) mRNA expression levels under the GcB5-induced indicated that the observing genes were affected significantly by expression of GcB5 gene in E. coli. The transcript level of observing genes such as grpE, ompA, oxyR, pspA, cysK, ompF, clpX, hslU, pnp, minD, yeaD, tolC and dacA were increased prominently in E. coli BL21/pET28a:GcB5 at 1.5h compared to those in control E. coli BL21/pET28a. pspA was only expression in E. coli BL21/pET28a:GcB5, but was not almost expression in CK. Those results or tendency are consistent with the results of 2-DE. However, the transcript levels of three genes (ftsZ, mreB and oxyR) were almost unchanged in E. coli BL21/pET28a:GcB5 compared to those in control E. coli BL21/pET28a.